A zinc-doped carbon quantum dot from orange peel and its application in alleviating abiotic stress in honeysuckle berries.

Abstract

This invention belongs to the field of agricultural nanomaterials and heavy metal pollution remediation technology, specifically relating to a zinc-doped carbon quantum dot (Zn-CDs) from orange peel and its application in alleviating abiotic stress in honeysuckle berries. The Zn-CDs are prepared by hydrothermal synthesis using orange peel powder and zinc chloride as raw materials. This invention also discloses the application of these Zn-CDs in alleviating abiotic stress in honeysuckle berries. Applied to honeysuckle berry plants under salt and / or cadmium stress via foliar spraying, it significantly alleviates cadmium and salt stress by reducing heavy metal absorption, activating the antioxidant system, improving photosynthetic efficiency, and regulating hormone and signaling pathway genes, thereby restoring plant growth. This provides a green nano-resistance agent for the safe production of honeysuckle berries in heavy metal / salt-affected areas, while simultaneously realizing the resource utilization of orange peel.

Description

Technical Field

[0001] This invention belongs to the field of agricultural nanomaterials and heavy metal pollution remediation technology, specifically relating to a zinc-doped carbon quantum dot for orange peel and its application in alleviating abiotic stress in honeysuckle berries. Background Technology

[0002] With the rapid development of industrialization and agricultural modernization, heavy metal pollution has become a global environmental problem, posing a serious threat to ecosystems and human health. Cadmium (Cd), a common heavy metal pollutant, is highly toxic and persistent, capable of entering the food chain through the soil-plant system and ultimately harming human health. Cadmium stress not only affects plant growth and development but also leads to the excessive accumulation of reactive oxygen species (ROS) in plants, triggering oxidative stress, which in turn damages cell membrane structure, inhibits photosynthesis, and interferes with enzyme activity. Therefore, how to effectively mitigate the toxic effects of cadmium stress on plants has become a research hotspot in the fields of agriculture and environmental science.

[0003] Honeysuckle berries, scientifically known as *Lonicera caerulea* L., are perennial deciduous shrubs belonging to the genus *Lonicera* in the family Caprifoliaceae. They are small berries unique to cold regions. Honeysuckle berries contain high levels of anthocyanins, vitamins, amino acids, and polyphenols, possessing physiological functions such as anti-aging, antioxidant, lipid-lowering, vision protection, cardiovascular disease prevention, and anti-cancer and anti-tumor effects. As a berry plant with high economic and nutritional value, honeysuckle berries have received widespread attention in recent years. Honeysuckle berries are distributed in North my country, Northeast China, and Xinjiang, with the main cultivation area being Northeast my country, including the Greater and Lesser Khingan Mountains and Changbai Mountains. However, heavy metal pollution is particularly severe in the old industrial bases of Northeast China compared to other regions. Therefore, honeysuckle berries are susceptible to abiotic stress during their growth. Soil salinization and heavy metal pollution are significant environmental problems restricting agricultural production and ecological restoration, posing a serious threat to the growth of honeysuckle berries and other economic crops. Therefore, exploring effective methods to alleviate salt and cadmium stress is of great significance for improving the yield and quality of honeysuckle berries.

[0004] Carbon quantum dots (CDs) are novel carbon-based nanomaterials with a particle size of less than 10 nm and exhibiting significant photofluorescence effects. In recent years, the applications of carbon quantum dots in biomedicine, environmental remediation, and agriculture have gradually attracted attention. CDs possess numerous advantages, including excellent optical properties, good water solubility, low toxicity, environmental friendliness, biocompatibility, wide availability of raw materials, and low preparation cost. Furthermore, CDs possess abundant surface functional groups, exhibiting excellent photophysical and photochemical properties and demonstrating great application potential in enhancing crop stress resistance and promoting crop growth. However, existing research on carbon quantum dots for alleviating salt and cadmium stress mainly focuses on enhancing antioxidant enzyme activity and physical adsorption and immobilization, lacking systematic regulation of plant signaling pathways. Summary of the Invention

[0005] To address the aforementioned problems in the existing technology, the present invention aims to provide a zinc-doped carbon quantum dot for orange peel and its application in mitigating abiotic stress in honeysuckle berries.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a zinc-doped carbon quantum dot from orange peel for use in treating abiotic stress in honeysuckle berries. The zinc-doped carbon quantum dot from orange peel is obtained by hydrothermal carbonization using orange peel powder and zinc chloride as raw materials.

[0007] The zinc-doped carbon quantum dots in orange peel of this invention can activate the expression of key genes in four major functional modules of honeysuckle fruit: signal transduction, secondary metabolism, photosynthesis, and antioxidant system, thereby enhancing the physiological adaptability of honeysuckle fruit to salt stress and / or cadmium stress, and thus reducing cell damage caused by salt stress and / or cadmium stress.

[0008] Furthermore, the mass ratio of the orange peel powder to zinc chloride is 4~5:3.

[0009] Furthermore, the solvent in the hydrothermal method is water, and the mass-to-volume ratio of the orange peel powder to water is 4~5g:600~800mL.

[0010] Furthermore, hydrothermal carbonization involves a hydrothermal reaction at 190~210℃ for 10~12 hours.

[0011] Furthermore, after the hydrothermal reaction is completed, impurities are removed by filtration, and then purified by dialysis to obtain the orange peel zinc-doped carbon quantum dots.

[0012] Furthermore, the molecular weight cutoff for the dialysis is 900 Da to 1100 Da.

[0013] Furthermore, the orange peel zinc-doped carbon quantum dots have a particle size range of 2.13-3.36 nm.

[0014] Secondly, the present invention provides the application of the zinc-doped carbon quantum dots from orange peel in the abiotic stress of honeysuckle berries, wherein the abiotic stress is salt stress and / or cadmium stress.

[0015] Furthermore, a solution of zinc-doped carbon quantum dots from orange peel with a concentration of 800-1200 mg / L was applied to honeysuckle berry plants under salt stress and / or cadmium stress by foliar spraying.

[0016] The beneficial effects of this invention are: This invention uses agricultural waste orange peel as a carbon source and combines it with ZnCl2 to prepare Zn-CDs via a one-step hydrothermal method. The raw materials are readily available and inexpensive, realizing the resource utilization of agricultural waste. The preparation process does not involve the addition of toxic or harmful reagents, making it environmentally friendly. The process is simple and easy to scale up.

[0017] The orange peel Zn-CDs prepared by this invention have uniform particle size, good water solubility, excellent fluorescence properties, rich hydrophilic functional groups on the surface, and appropriate zinc doping amount, which can effectively enter the leaves of honeysuckle fruit to exert their effects. They also have good biocompatibility and no obvious toxicity.

[0018] This invention applies Zn-CDs from orange peel to alleviate cadmium stress in honeysuckle berries. The foliar spraying method is simple to operate and has a high utilization rate. It can significantly alleviate the toxicity of salt stress and / or cadmium stress on honeysuckle berries and improve the growth capacity, yield and quality of honeysuckle berries in heavy metal / saline areas through multiple synergistic effects, such as increasing biomass, optimizing root system, enhancing photosynthetic efficiency, activating antioxidant system, reducing cadmium absorption, and regulating the expression of key genes. Attached Figure Description

[0019] Figure 1 Characterization of Zn-CDs. (a) TEM image and particle size distribution of Zn-CDs, scale bar = 10 nm. (b) UV-Vis absorption spectrum of Zn-CDs (inset: Zn-CDs solution under sunlight and 365 nm UV light). (c) Fluorescence emission spectrum of Zn-CDs solution at different excitation wavelengths (315~475 nm). (d) FTIR spectrum of Zn-CDs. (e) XRD diffraction pattern of Zn-CDs. (f) XPS spectrum of Zn-CDs. (g) High-resolution XPS fitted spectrum of C1s. (h) High-resolution XPS fitted spectrum of N1s. (i) High-resolution XPS fitted spectrum of iO1s. (j) High-resolution XPS fitted spectrum of Zn2p.

[0020] Figure 2 Laser confocal microscopy images of Zn-CDs in chloroplasts of honeysuckle berries under cadmium stress. (Green fluorescence represents the fluorescence signal of Zn-CDs, and red fluorescence represents the fluorescence signal of chloroplasts).

[0021] Figure 3 The effects of different treatments on the growth of *Honeysuckle berries* under cadmium stress and on cadmium accumulation in *Honeysuckle berries* leaves. (a) Phenotypic observation of *Honeysuckle berries* plants (from left to right: CK, Zn-CDs, CdCl2+Zn-CDs, and CdCl2 treatment groups). (b) Plant height of *Honeysuckle berries* plants. (c) Stem diameter. (d) Fresh weight of *Honeysuckle berries* plants. (e) Dry weight of *Honeysuckle berries* plants. (f) Effect of cadmium content in *Honeysuckle berries* plant leaves. Data are mean ± SE. Different letters indicate significant differences between treatments (p<0.05). The same letter indicates no significant difference.

[0022] Figure 4 The effects of Zn-CDs on root traits of honeysuckle berry plants under cadmium stress. (a) Root morphology of honeysuckle berry plants treated with CK, Zn-CDs, CdCl2+Zn-CDs, and CdCl2, respectively. (b) Total root length. (c) Projected area. (d) Specific surface area. (e) Volume. (f) Mean root diameter. Data are presented as mean ± SE. Different letters indicate significant differences between treatments (p < 0.05). The same letter indicates no significant difference.

[0023] Figure 5 Effects of Zn-CDs on photosynthesis in honeysuckle berry plants under cadmium stress. (a) Net photosynthetic rate (Pn). (b) Stomatal conductance (Cond). (c) Transpiration rate (Trmmol). (d) Intercellular CO2 concentration (Ci). (e) Fv / Fm. (f) Total chlorophyll content. (g) Chlorophyll a content. (h) Effect of chlorophyll b content. Data are mean ± SE. Different letters indicate significant differences between treatments (p<0.05). The same letter indicates no significant difference.

[0024] Figure 6 The effects of Zn-CDs on the antioxidant defense system of honeysuckle berry plants under cadmium stress. (a) SOD activity. (b) POD activity. (c) CAT activity. (d) MDA content. (e) H2O2 content. (f) O 2- Content. (g) Confocal imaging of DCF (mainly indicating H2O2). (h) DCF fluorescence intensity statistics. (i) DHE (mainly indicating O2). 2- (j) Confocal imaging. DCF fluorescence intensity statistics. Data are mean ± SE. Different letters indicate significant differences between different treatments (p<0.05). The same letter indicates no significant difference.

[0025] Figure 7 DEGs in key pathways of honeysuckle fruit treated with Zn-CDs under cadmium stress.

[0026] Figure 8qRT-PCR validation of transcriptome data from Zn-CDs-treated honeysuckle berries under cadmium stress. The bars (left y-axis) in the figure represent the relative expression levels from qRT-PCR, and the line (right y-axis) shows the FPKM values ​​from RNA-seq data. Data are expressed as mean ± standard deviation. (a) to (i) represent LC_011075, LC_022873, LC_014642, LC_016476, LC_004095, LC_030554, LC_019949, LC_038659, and LC_020543, respectively.

[0027] Figure 9 This is a laser confocal image of Zn-CDs in the chloroplasts of *Honeysuckle berries* leaves under salt stress. (Green fluorescence represents the fluorescence signal of Zn-CDs, and red fluorescence represents the fluorescence signal of chloroplasts.)

[0028] Figure 10 The effect of Zn-CDs on the growth of honeysuckle berries under salt stress. (a) Phenotypic observation of honeysuckle berry plants (from left to right: CK, Zn-CDs, NaCl+Zn-CDs, and NaCl treatment groups). (b) Plant height of honeysuckle berry plants. (c) Stem diameter. (d) Fresh weight of honeysuckle berry plants. (e) Dry weight of honeysuckle berry plants.

[0029] Figure 11 The effects of Zn-CDs on root traits of honeysuckle berry plants under salt stress. (a) Root morphology of honeysuckle berry plants under CK, Zn-CDs, NaCl + Zn-CDs, and NaCl treatments, respectively. (b) Total root length. (c) Projected area. (d) Specific surface area. (e) Volume. (f) Mean root diameter. Data are presented as mean ± SE. Different letters indicate significant differences between treatments (p < 0.05). The same letter indicates no significant difference.

[0030] Figure 12 The effects of Zn-CDs on photosynthesis in honeysuckle berry plants under salt stress were investigated. (a) Net photosynthetic rate (Pn). (b) Stomatal conductance (Cond). (c) Transpiration rate (Trmmol). (d) Intercellular CO2 concentration (Ci). (e) Fv / Fm. (f) Total chlorophyll content. (g) Chlorophyll a content. and (h) Chlorophyll b content. Data are mean ± SE. Different letters indicate significant differences between treatments (p < 0.05). The same letter indicates no significant difference.

[0031] Figure 13 The effect of Zn-CDs on chlorophyll content in honeysuckle berry plants under salt stress. (a) Total chlorophyll content. (b) Chlorophyll a content. (c) Chlorophyll b content.

[0032] Figure 14 The effects of Zn-CDs on MDA and reactive oxygen species (ROS) content in honeysuckle berry plants under salt stress. (a) MDA content. (b) H2O2 content. (c) O2 content. 2- content.

[0033] Figure 15 The effects of Zn-CDs on the antioxidant defense system of honeysuckle berries under salt stress. (a) SOD activity. (b) POD activity. (c) CAT activity.

[0034] Figure 16 DEGs on key pathways in honeysuckle fruit treated with Zn-CDs under salt stress.

[0035] Figure 17 qRT-PCR validation of transcriptome data from Zn-CDs-treated honeysuckle plants under salt stress. (a) to (i) are LC_011075, LC_022873, LC_014642, LC_016476, LC_004095, LC_030554, LC_019949, LC_038659, and LC_020543, respectively. Detailed Implementation

[0036] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0037] Example 1: Synthesis and characterization of orange peel zinc-doped carbon quantum dots (Zn-CDs).

[0038] 1. Method The preparation method of orange peel Zn-CDs is a hydrothermal method. The specific synthesis and preparation method is as follows: 2.00 g of orange peel powder and 1.50 g of ZnCl2 were added to 300 mL of deionized water and mixed thoroughly. Then, the mixture was heated at 200 °C for 10 h in a PTFE-lined stainless steel autoclave. After the solution cooled to room temperature, large particles were removed using a 0.22 μm organic phase needle filter, followed by further purification via dialysis in a 1000 Da dialysis bag. Finally, the purified solution was freeze-dried for 48 h to obtain orange peel Zn-CDs, which were then prepared into a 1000 mg / L carbon quantum dot solution using deionized water for experimental treatment.

[0039] The morphology and particle size of Zn-CDs were detected and quantified using transmission electron microscopy (TEM) (JEOL JEM-2100F, Japan). UV-Vis spectroscopy was performed using a nanophotometer (N50). The emission spectrum of PL was recorded using a fluorescence spectrophotometer. Fourier transform infrared spectroscopy (FTIR) was performed using a Fourier transform infrared absorption spectrometer (Thermo Fisher Scientific, USA, IS50) to determine the presence of functional groups. The crystal properties of Zn-CDs were characterized using X-ray diffraction (XRD) (Bruker D8 Advance, Germany). The surface chemical composition and electronic structure of Zn-CDs were further analyzed using X-ray photoelectron spectroscopy (XPS) (Thermo Fisher Scientific K-Alpha, USA).

[0040] 2. Results Transmission electron microscopy images show that the carbon dots prepared from orange peel are spherical nanoparticles, and the Zn-CDs are monodisperse in different shapes, all less than 10 nm in the quantum range. Figure 1 Image a shows the particle size distribution of Zn-CDs, ranging from 2.13 to 3.36 nm, with an average diameter of 2.58 nm. High-resolution transmission images of orange peel Zn-CDs reveal distinct lattice fringes, with a crystal planar spacing of approximately 0.207 nm. Ultraviolet-Vis absorption spectroscopy (UV-Vis) is used to further analyze the particle size distribution. Figure 1 (b) An absorption band was observed at approximately 280 nm, which is attributed to the π→π* transition of the aromatic structure containing C=O and CN. The Zn-CDs solution emitted blue fluorescence under 365 nm UV light; this instantaneous color change under UV light is an inherent characteristic of carbon dots, confirming the synthesis of Zn-CDs. The fluorescence properties of Zn-CDs were determined using fluorescence spectroscopy. Figure 1 Figure c shows the excitation-dependent fluorescence emission of Zn-CDs. A significant and uniform color shift in the emission peak occurred as the excitation wavelength increased from 315 nm to 475 nm. The position and intensity of the fluorescence emission peak varied with the excitation wavelength, indicating that fluorescence emission is excitation-dependent. Furthermore, the maximum excitation wavelength for Zn-CDs was 375 nm, at which the maximum fluorescence intensity was approximately 450 nm.

[0041] To determine the crystal form of Zn-CDs, XRD analysis was performed. The XRD pattern showed a broad peak centered at 27.92°, confirming the presence of the Zn-CDs crystalline phase. Figure 1 (e). The chemical composition and surface state of Zn-CDs were further analyzed using Fourier transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS). FTIR spectroscopy showed that in the 3200-3600 cm⁻¹ range... -1A characteristic broad absorption band was observed at 1592, 1425, and 1214 cm⁻¹, which is due to the stretching vibration of OH groups. -1 The peaks at these points correspond to the stretching vibrations of C=C, CN, and CO, respectively. Figure 1 (d). The surface functional groups of Zn-CDs were further detected by XPS analysis. Zn-CDs are mainly composed of C (284.71 eV, At%=62.54), N (400.4 eV, At%=2.28), O (532.56 eV, At%=26.50), and Zn (1022.57 eV, At%=8.68) elements (Fig. 1f). C1s spectra showed that Zn-CDs contained three peaks: O=CO (288.9 eV), CO / N (286.3 eV), and CC (284.7 eV). Figure 1 The N1s spectrum involves two peaks: NO (401.8 eV) and NCN (400.2 eV). Figure 1 The O1s spectrum can be fitted to two peaks: CO (532.9 eV) and oxygen vacancy (532.2 eV). Figure 1 The Zn2p spectrum fits to two peaks: Zn2p1 / 2 (1045.5 eV) and Zn2p3 / 2 (1022.5 eV). Figure 1 (j). FTIR and XPS spectroscopy revealed that the prepared Zn-CDs contained abundant hydrophilic functional groups such as amino groups or oxygen-containing groups, proving that Zn-CDs can exist stably in aqueous solution.

[0042] Example 2: Study on the mitigation of salt and cadmium stress in honeysuckle berry plants by zinc-doped fluorescent carbon dots 1. Method 1.1 Experimental Materials and Treatment The experiment was conducted at the small berry cultivation resource nursery of the Horticulture Station of Northeast Agricultural University. Using two-year-old cuttings of the 'Wulan' variety of honeysuckle berries as experimental material, the effects of zinc-doped fluorescent carbon dots (Zn-CDs) on alleviating the growth of honeysuckle berry plants under salt and cadmium stress were studied.

[0043] After two weeks of growth under identical environmental conditions, two-year-old honeysuckle cuttings that were free from pests and diseases and exhibited good and uniform growth were selected for salt stress (NaCl concentration of 200 mmol / L) and cadmium stress (CdCl2 concentration of 50 mg / kg). Once the plants exhibited the desired phenotype, they were treated with a foliar spray of a 1000 mg / L carbon dot solution (seedlings not treated with carbon dot solution were sprayed with deionized water under consistent control conditions). The salt stress experiment included four treatments: control group (CK), Zn-CDs alone (Zn-CDs), Zn-CDs and NaCl combined treatment group (Zn-CDs+NaCl), and NaCl stress treatment group (NaCl). The cadmium stress experiment also included four treatments: control group (CK), Zn-CDs alone (Zn-CDs), Zn-CDs and CdCl2 combined treatment group (Zn-CDs+CdCl2), and CdCl2 stress treatment group (CdCl2). Each treatment had 5 replicates, and all plants were placed in a small greenhouse.

[0044] Each plant was sprayed with 20 mL of the treatment solution only on day 1, with each spraying continuing until water droplets appeared on the leaves. This process was repeated every 3 days at the same time (days 5, 9, 13, 17, 21, and 25), for a total of 28 days. On day 28 of treatment, functional leaves and fine roots from each group of plants were collected. A portion was used for physiological and biochemical index determination, while the other portion was flash-frozen in liquid nitrogen and stored at -80°C for further index determination.

[0045] 1.2 Measurement Indicators 1.2.1 Measurement of physiological indicators (1) Plant height measurement: The height of seedlings was measured using the ruler method, from the substrate surface to the apical growing point of the seedling. The height of cuttings was measured using the tape measure method, from the soil surface to the tip of the highest branch.

[0046] (2) Stem diameter measurement: The stem diameter was measured using a vernier caliper at a distance of 1 cm from the substrate surface. Each sample was measured 3 times and the average value was taken.

[0047] (3) Dry weight and fresh weight determination: The plants were rinsed with distilled water, and the surface moisture was absorbed with filter paper. The fresh weight was weighed immediately. The samples were then placed in an oven at 105℃ for 30 min to fix the green color, and then dried at 75℃ until constant weight was achieved. After cooling, the dry weight was weighed. All weighing operations were performed using an electronic analytical balance, and each sample was measured three times.

[0048] 1.2.2 Root system trait indicators The roots were scanned using a CrystalScanMaker i800Plus flatbed scanner and analyzed using the Wanshen LA-S root analysis system. Five parameters were measured and analyzed: total root length, projected area, specific surface area, volume, and average root diameter. Each root system was measured three times, and the average value was taken as the final result.

[0049] 1.2.3 Measurement of photosynthetic indicators Photosynthetic characteristics were measured using a Li-6400 portable photosynthesis meter (Li-Cor6400, USA). The measured parameters included net photosynthetic rate (Pn), stomatal conductance (Cond), intercellular CO2 concentration (Ci), and transpiration rate (Tr). Measurements were taken on a clear, windless morning from 5:00 to 7:00, with a light intensity of 1000 μmol·m⁻²·s⁻¹. Three seedlings of uniform growth were selected for each treatment. From each seedling, three robust, fully expanded leaves of uniform position were selected for measurement. Each leaf was measured three times, and the average value was taken as the final result.

[0050] Chlorophyll fluorescence parameters were measured using a Handy PEA plus portable plant efficiency analyzer (Hansatech Instruments Ltd., UK). Healthy, fully expanded functional leaves from *Honeysuckle berries* cuttings were selected for measurement. Three seedlings were randomly selected for each treatment, and three leaves were measured from each seedling. Each leaf was measured three times, resulting in a total of nine measurements for each treatment. Leaves were allowed to dark adapt for 20 minutes before measurement to allow photosystem II (PSII) to reach redox equilibrium.

[0051] 1.2.4 Determination of Reactive Oxygen Species and Malondialdehyde (MDA) Content Hydrogen peroxide (H2O2), superoxide anion (O2O2) 2- The assay kits for malondialdehyde (MDA) and malondialdehyde (MDA) were purchased from Beijing Solarbio Biotechnology Co., Ltd., and the procedures were performed according to the kit instructions.

[0052] 1.2.5 Antioxidant enzyme assay The superoxide dismutase (SOD) and peroxidase (POD) activity assay kits were purchased from Beijing Solarbio Biotechnology Co., Ltd. The catalase (CAT) activity assay kit was purchased from Nanjing Jiancheng Biotechnology Institute.

[0053] 1.2.6 Determination of Cadmium Ion Content The cadmium ion content in honeysuckle berry leaves was determined using an inductively coupled plasma atomic emission spectrometer (ICAP-PRO). The dried leaves were ground into a fine powder, and the plant sample was digested with concentrated HNO3. Instrument parameters: emission power: 1350 W; cooling gas: 14 L / min; combustion gas: 0.75 L / min; nebulizing gas: 0.76 L / min. The calculation formula is as follows:

[0054] In the formula: m0: The mass of the sample used in the analysis, in grams; V0: The volume of the sample after digestion and final volume adjustment, in mL; f: Dilution factor; Co: The concentration of the element in the test solution, in mg / L; C1: Elemental concentration of the original sample digest solution, in mg / L. C1 (mg / L) = CO (mg / L) * f * V0; Cx: The final test result of the element being measured, in mg / kg.

[0055] 1.2.7 Laser confocal imaging Co-localization of Zn-CDs in honeysuckle berry leaves. After applying Zn-CDs to the leaves, the leaves were incubated in the dark for 3 hours. Small round slides (5 mm in diameter) were then prepared, placed face up on a glass slide, and pressed. The samples were imaged using a laser confocal microscope (Olympus Corporation, Japan). The parameters were set as follows: 514 nm laser excitation, PMT1, 550 nm–615 nm (receiving Zn-CD fluorescence); PMT2, 700 nm–750 nm (receiving chloroplast fluorescence).

[0056] In vivo imaging of reactive oxygen species. After 28 days of cadmium stress treatment, small circular slides (5 mm in diameter) were taken from the first and second true leaves, respectively. Three to five small holes were made in each slide using pointed tweezers. The slides were then incubated with 25 μM 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA, primarily indicating H2O2) and 10 μM dihydroethidium (DHE, primarily indicating O2), respectively. 2-Both probes were diluted with 10 mM TES buffer (pH 7.5). After incubation for 30 min, the discs were rinsed three times with TES buffer, placed face up on a glass slide, and pressed. The samples were imaged using a laser confocal microscope FV3000 (Olympus Corporation, Japan). The parameters were set as follows: 488 nm laser excitation, PMT1, 500 nm–600 nm (receiving fluorescence from DCF and DHE); PMT2, 700 nm–785 nm (receiving fluorescence from chloroplasts). Each treatment was performed in at least three replicates.

[0057] 1.2.8 Transcriptomics Sequencing and Data Analysis Transcriptional sequencing experiments were performed on honeysuckle berry leaves collected after 28 days of treatment, including the control group (CK), the Zn-CDs-only treatment group (Zn-CDs), the Zn-CDs and NaCl co-treatment group (Zn-CDs+NaCl), the NaCl stress treatment group (NaCl), the Zn-CDs and CdCl2 co-treatment group (Zn-CDs+CdCl2), and the CdCl2 stress treatment group (CdCl2). Each treatment was performed in triplicate.

[0058] Transcriptomic analysis was performed on honeysuckle berry leaves under each treatment, with three independent biological replicates for each group. Transcriptomic sequencing was performed by BioChem. The transcriptomic sequencing experimental procedure included RNA extraction, RNA detection, library construction, and sequencing. RNA from the total samples was isolated and purified using TRIzol (thermofisher, 15596018) according to the manufacturer's operating protocol.

[0059] Then, the quantity and purity of total RNA were quality controlled using a NanoDrop ND-1000 (NanoDrop, Wilmington, DE, USA), and RNA integrity was tested using a Bioanalyzer 2100 (Agilent, CA, USA); concentration >50 ng / μL, RIN value >7.0, and total RNA >1 μg met the requirements for downstream experiments. The next step was library construction. After the sample passed the quality control tests, library construction and quality control were performed. Finally, Illumina Novaseq was used for analysis. TM The 6000 genome was sequenced using standard procedures with paired ends in PE150 mode. After obtaining the sequencing data, it was first filtered to obtain high-quality sequencing data (CleanData). This high-quality sequencing data was then aligned to the reference genome of the species in this project, and gene expression quantification, GSEA, differential gene analysis, and enrichment analysis were performed. The differential gene screening criteria were FoldChange ≥ 2 and FDR < 0.01.

[0060] 1.2.9 Real-time quantitative PCR (qRT-PCR) To ensure the reliability of the transcriptome, nine genes were randomly selected for real-time quantitative PCR (RT-qPCR) analysis, using three independent biological replicates. Specific quantitative primers were designed using Primer 5.0 software based on the *Honeysuckle berries* genome reference sequence, with β-Actin as the internal control gene. Total RNA was extracted from FAZ samples using the EASYspinPlus Complex Plant RNA Kit (Aidlab) according to the manufacturer's instructions. cDNA was synthesized from total RNA using the ReverTra Ace qPCR RTMasterMix (TOYOBO) according to the manufacturer's instructions. qRT-PCR detection was performed using a SYBR® Green Realtime PCR MasterMix (TOYOBO) and a qTOWER3 real-time PCR instrument (Analytik Jena). The qRT-PCR program used the following parameters: 95°C denaturation for 15 s, 55°C annealing for 20 s, 72°C extension for 15 s, 40 cycles, and termination at 4°C. The relative gene expression levels were calculated using the 2-ΔΔCT method. We used Excel, GraphPad Prism, and TBtools software for data analysis and graphing.

[0061] 1.2.10 Data Analysis All experimental data are expressed as mean ± standard deviation (SD), with three or more replicates per treatment. Statistical analysis was performed using OriginPro 2024 software, with P < 0.05 considered statistically significant. One-way ANOVA was performed using SPSS software to assess differences between treatments.

[0062] 2. Results 2.1 Study on the use of Zn-CDs to alleviate cadmium stress in honeysuckle berry plants 2.1.1 Localization of Zn-CDs in honeysuckle berry leaves After 3 hours of low light treatment, no fluorescence signal of Zn-CDs was found in the leaves of both the control group and the cadmium-stressed treatment, while a significant signal was found in the leaves treated with Zn-CDs. This result indicates that Zn-CDs play a role in the entry of Zn-CDs into the leaves of honeysuckle berries. Figure 2 ).

[0063] 2.1.2 Zn-CDs alleviate cadmium stress in honeysuckle berries and reduce cadmium accumulation in honeysuckle berry leaves. like Figure 3As shown in Figure a, spraying Zn-CDs effectively promoted the growth of honeysuckle berry plants, demonstrating a significant growth-promoting effect. Compared with the control, the honeysuckle berry plants sprayed with Zn-CDs showed vigorous leaf growth, and significantly increased plant height (6.7%), stem diameter (5.6%), fresh weight (94.6%), and dry weight (78.9%). Figure 3 (be). After applying 50 mg / kg cadmium stress, the growth of honeysuckle berry plants was significantly inhibited, and the leaf margins turned yellow. However, Zn-CDs effectively alleviated this growth inhibition, increasing plant height, stem diameter, fresh weight, and dry weight by 25.1%, 18.4%, 43.5%, and 40.9%, respectively. Figure 3 These growth benefits may be due to the reduced toxicity of Cd immobilization in plants, as well as the direct growth-stimulating effect of Zn-CDs on plants. These results indicate that Zn-CDs can significantly increase the biomass accumulation of honeysuckle berries and have broad application prospects in promoting crop growth.

[0064] like Figure 3 As shown in f, the Cd content in the leaves of honeysuckle berry plants under cadmium stress... 2+ The content was significantly higher than that of the control group, and Zn-CDs could effectively alleviate cadmium accumulation in plant leaves. Compared with the CdCl2 treatment group, Cd... 2+ The content decreased by 43.3%. This indicates that CDs can act as a potential promoter, encouraging hyperaccumulating plants to absorb more heavy metals, thereby improving phytoremediation efficiency. Notably, the Cd content decreased under Zn-CDs treatment conditions. 2+ The concentration was higher than the control group, possibly because the carbon dots used in the experiment were zinc-doped. Cadmium, as a typical heavy metal, has a low abundance in the Earth's crust, and its natural occurrence is often accompanied by large amounts of zinc. The two are prone to forming symbiotic relationships during geochemical processes. During plant absorption, Cd... 2+ Zn can be used 2+ With Ca 2+ The low specificity of ion channels allows Cd to enter plant cells—specifically, Cd. 2+ By specifically binding to zinc and calcium transporters, transmembrane transport in root epidermal cells is accomplished via the symplast pathway.

[0065] 2.1.3 Zn-CDs alleviate the effects of cadmium stress on the root system of honeysuckle berries Under cadmium stress, Zn-CDs treatment significantly improved and even enhanced the root morphology of honeysuckle berry plants. Figure 4 Compared with the control group, Zn-CDs treatment significantly increased the total root length, projected area, specific surface area, and volume, by 39.84%, 36.03%, 37.19%, and 24.59%, respectively. Figure 4(bf). Under cadmium stress, Zn-CDs treatment effectively alleviated the root morphological traits of honeysuckle berry plants, increasing total root length, projected area, specific surface area, volume, and average root diameter by 10.82%, 10.86%, 8.1%, 52.94%, and 9.19%, respectively. Figure 4 (bf). In addition, although Zn-CDs treatment had a mitigating and promoting effect on the average root diameter, it had no significant effect.

[0066] 2.1.4 Zn-CDs enhance photosynthesis in honeysuckle berry plants The results showed that the net photosynthetic rate (Pn) (19.8%), stomatal conductance (Cond) (73.9%), and transpiration rate (Tr) (4.4%) of the honeysuckle berry leaves in the Zn-CDs treatment group were significantly higher than those in the control group. Figure 5 The intercellular CO2 concentration (Ci) decreased by 15.1% (ac). Figure 5 (d). Furthermore, foliar spraying with Zn-CDs reduced the harmful effects of cadmium stress on the photosynthesis of honeysuckle berry plants, increasing Pn, Cond, and Tr by 67.2%, 61.5%, and 40.9%, respectively, while decreasing Ci by 11.2% (d). Figure 5 The Fv / Fm ratio of honeysuckle berry plants treated with Zn-CDs was not significantly different from that of the control, indicating that Zn-CDs did not cause oxidative stress in honeysuckle berry plants. Furthermore, under cadmium stress, the Fv / Fm ratio of the Zn-CDs-treated group (CdCl2+Zn-CDs) was significantly higher than that of the CdCl2-treated group (5.0%), demonstrating that Zn-CDs can alleviate cadmium stress by regulating chlorophyll fluorescence parameters and improving photosynthesis in honeysuckle berry plants. Figure 5 (e).

[0067] Compared with the control group, the total chlorophyll, chlorophyll a, and chlorophyll b contents of the Zn-CDs treatment group increased by 42.0%, 47.5%, and 31.7%, respectively. The total chlorophyll (50.2%), chlorophyll a (71.3%), and chlorophyll b contents (21.8%) of the CdCl2+Zn-CDs treatment group were significantly higher than those of the CdCl2 treatment group. Figure 5 The results showed that Zn-CDs could increase the chlorophyll content of honeysuckle berries and effectively alleviate the inhibitory effect of cadmium stress on chlorophyll synthesis.

[0068] 2.1.5 Zn-CDs enhance the antioxidant defense system of honeysuckle berry plants Under cadmium stress, the activities of SOD (79.3%), POD (21.8%), and CAT (7.4%) in honeysuckle berry plants sprayed with Zn-CDs were significantly higher than those in the untreated group. Figure 6(ac), MDA, H2O2, O 2- The contents decreased by 17.3%, 62.1%, and 36.8%, respectively. These results indicate that Zn-CDs can significantly enhance the activity of antioxidant enzymes and alleviate oxidative damage caused by cadmium stress. Under no-stress conditions, Zn-CDs treatment also increased the activities of SOD (19.8%), POD (20.7%), and CAT (28.4%), and decreased the contents of MDA and H2O2. Figure 6 (df), but O 2- The higher content compared to the control group may be due to the mild oxidation signal induced by Zn-CDs, which temporarily caused the ROS generation rate to exceed the scavenging rate, resulting in a temporary accumulation of O2-. Overall, Zn-CDs can activate the defense system by regulating ROS signaling under non-stress conditions, while effectively enhancing antioxidant defense capabilities under Cd stress, thereby alleviating the damage caused by oxidative stress.

[0069] The results of laser confocal imaging further validated the above conclusions. Figure 6 Zn-CDs helped maintain ROS homeostasis in honeysuckle berries. In leaves treated with Zn-CDs under cadmium stress, DCF fluorescence intensity was significantly weaker than in the cadmium-treated group but stronger than in the control group, and quantitative fluorescence results showed the same trend. DHE staining results also showed a similar pattern. Zn-CDs treatment significantly reduced DHE fluorescence intensity under Cd stress, indicating that Zn-CDs can effectively reduce the accumulation of superoxide anions. These results suggest that the application of Zn-CDs helps maintain ROS homeostasis in honeysuckle berry plants and enhances their antioxidant defense system. In the ROS scavenging system, SOD is the main defense enzyme, followed by POD and CAT. This indicates that Zn-CDs can alleviate oxidative damage caused by Cd stress by activating the plant's endogenous antioxidant defense response and promoting the synergistic effect of the antioxidant enzyme system.

[0070] 2.1.6 Screening of genes related to key pathways in honeysuckle fruit after Zn-CDs application under cadmium stress Under cadmium stress conditions, the expression profile analysis of key genes in honeysuckle fruit showed that (…) Figure 7The main functions of Zn-CDs are concentrated in four major biological processes: signal transduction, secondary metabolism, photosynthesis, and the antioxidant system. Specifically, differentially expressed genes (DEGs) related to signal transduction are mainly distributed in the MAPK signaling pathway – plant and planthormone signal transduction pathways; genes related to secondary metabolism are concentrated in the cutin, suberine and wax biosynthesis, and phenylpropanoid biosynthesis pathways; DEGs related to photosynthesis are significantly altered in the oxidative phosphorylation and photosynthesis pathways; and genes involved in the antioxidant system are mainly enriched in the glutathione metabolism and peroxisome-related pathways. The study observed that in the single CdCl2 treatment group, most DEGs in the above pathways were significantly downregulated; while under Zn-CDs synergistic treatment, the expression levels of these genes all showed a significant rebound. This result suggests that Zn-CDs may enhance the physiological adaptation of honeysuckle fruit to cadmium stress by activating the gene expression of the above key metabolic and response pathways, thereby alleviating CdCl2-induced cell damage.

[0071] 2.2.7 qRT-PCR Validation To verify the reliability of the transcriptome analysis data, nine annotated single genes were selected for qRT-PCR analysis. These genes are closely related to pathways such as plant hormone signal transduction, Starch and sucrose metabolism, MAPK signaling pathway (plant), Glycerolipid metabolism, and Glutathione metabolism. After validation, the expression patterns of the nine genes showed ( Figure 8 The qRT-PCR results and transcriptome results highly overlapped, indicating that the differential expression results from transcriptome sequencing analysis have high reliability.

[0072] 2.2 Study on the relief of salt stress in honeysuckle berry plants by Zn-CDs 2.2.1 Laser confocal imaging of Zn-CDs in honeysuckle berry leaves The experimental results showed (9) that after 3 hours of low-light cultivation, no obvious characteristic fluorescence signal of Zn-CDs was detected in the leaf samples of the control group and the salt stress treatment group; however, a significant and specific fluorescence distribution was observed in the leaves treated with Zn-CDs, indicating that Zn-CDs had successfully entered the leaf tissue and accumulated therein. This result confirms that Zn-CDs have the ability to cross the leaf surface barrier and enter the interior of honeysuckle berries, providing a basis for subsequent analysis of its physiological effects.

[0073] 2.2.2 Effects of Zn-CDs on the growth and development of honeysuckle berry plants After 28 days of treatment, the plant phenotypic observations are as follows: Figure 10 As shown in Figure a, Zn-CDs not only significantly promoted plant growth under normal conditions but also effectively alleviated growth inhibition caused by salt stress. Compared with the control, the honeysuckle berries sprayed with Zn-CDs exhibited vigorous leaf growth, and significantly increased plant height (6.7%), stem diameter (5.6%), fresh weight (94.6%), and dry weight (78.9%). Figure 10 (be). After applying 200 mmol / L salt stress, the growth of honeysuckle berry plants was significantly inhibited, and the leaf margins turned yellow. However, Zn-CDs effectively alleviated this growth inhibition, increasing plant height, stem diameter, fresh weight, and dry weight by 46.6%, 17.9%, 68.5%, and 42.9%, respectively. Figure 3-7 Furthermore, the fresh weight (52.4%) and dry weight (28.0%) in the NaCl+Zn-CDs treated group were significantly increased compared to the control group. Figure 10 The above results indicate that Zn-CDs can significantly increase the biomass accumulation of honeysuckle berries and have a positive promoting effect on their plant growth.

[0074] 2.2.3 Effects of Zn-CDs on root morphology of honeysuckle berry plants like Figure 11 As shown in Figure a, compared with the control, plants foliar-sprayed with Zn-CDs had more developed root systems and denser fibrous roots; the elongation of the taproot was strongly inhibited and the development of lateral roots was reduced under salt stress, while Zn-CDs effectively alleviated this damage. Specifically, compared with the control group, Zn-CDs treatment significantly increased the total root length, projected area, specific surface area, and volume, increasing by 39.84%, 36.03%, 37.19%, and 24.59%, respectively. Figure 11 (be). Under salt stress, Zn-CDs treatment effectively alleviated the root morphological traits of honeysuckle berry plants, increasing total root length, projected area, specific surface area, and average root diameter by 26.50%, 38.02%, 35.06%, and 6.80%, respectively. Figure 11Furthermore, although Zn-CDs treatment had a mitigating and promoting effect on the average root diameter, it had no significant effect (be). Figure 11 (e). The above results indicate that foliar spraying of Zn-CDs can improve or even enhance the root morphology and function of honeysuckle berry plants. This may be because Zn-CDs enter the leaves through stomata or epidermis and are transported to the roots through the vascular system, thereby indirectly regulating root growth and development by enhancing leaf photosynthesis and regulating root exudates.

[0075] 2.2.4 Effects of Zn-CDs on the photosynthetic characteristics of honeysuckle berry plants The net photosynthetic rate (Pn) (19.8%), stomatal conductance (Cond) (73.9%), and transpiration rate (Trmmol) (4.4%) of the honeysuckle berry leaves in the Zn-CDs treatment group were significantly higher than those in the control group. Figure 12 The intercellular CO2 concentration (Ci) was significantly reduced by 15.1% (ac). Figure 12 (d). Furthermore, foliar spraying with Zn-CDs reduced the harmful effects of salt stress on the photosynthesis of honeysuckle berry plants, increasing Pn, Cond, and Tr by 108.6%, 63.4%, and 82.6%, respectively, while decreasing Ci by 11.7% (d). Figure 12 These results indicate that Zn-CDs can effectively enhance the photosynthetic capacity of honeysuckle berry leaves, accompanied by increased water transpiration rate and improved leaf CO2 absorption and utilization efficiency.

[0076] The Fv / Fm ratio of honeysuckle berry plants treated with Zn-CDs was not significantly different from that of the control, indicating that Zn-CDs treatment did not have a significant adverse effect on PSII. Furthermore, under salt stress, the Fv / Fm ratio of the Zn-CDs-treated group (NaCl + Zn-CDs) was higher than that of the NaCl-treated group (4.5%), demonstrating that Zn-CDs can alleviate salt stress by regulating chlorophyll fluorescence parameters and improving photosynthesis in honeysuckle berry plants. Figure 12 (e).

[0077] Exogenous application of Zn-CDs significantly increased the chlorophyll content of honeysuckle berry leaves. Under normal growth conditions, compared with the control group, Zn-CDs treatment increased the total chlorophyll, chlorophyll a, and chlorophyll b contents by 42.0%, 47.5%, and 31.7%, respectively. Figure 13(ac). Notably, under 200 mmol / L NaCl stress, salt stress treatment led to a significant decrease in the content of various chlorophyll components; while after foliar spraying with Zn-CDs, the contents of total chlorophyll, chlorophyll a, and chlorophyll b significantly increased by 73.1%, 110.5%, and 31.1%, respectively. These results fully demonstrate that Zn-CDs can not only actively promote chlorophyll synthesis in honeysuckle berries under normal conditions, but also effectively alleviate the destructive effects of salt stress on chlorophyll metabolism and maintain a high pigment content level. This suggests that Zn-CDs treatment helps maintain a high chlorophyll level, which may be related to its ability to alleviate salt stress damage.

[0078] 2.2.5 Effects of Zn-CDs on the antioxidant defense system of honeysuckle berry plants Under normal growth conditions, compared with the control group, the contents of MDA and H2O2 in plants treated with Zn-CDs decreased by 12.5% ​​and 3.7%, respectively. Figure 14 (ab). Under salt stress, the levels of MDA (10.5%), H2O2 (61.3%), and O2 in honeysuckle berry plants sprayed with Zn-CDs were significantly higher. 2- The content (54.7%) was significantly lower than that of the untreated group ( Figure 14 (ac). However, under no-stress conditions, O 2- The content was higher than that of the control group ( Figure 14 (c) This may be because Zn-CDs induce a slight oxidation signal, causing the ROS generation rate to briefly exceed the scavenging rate, thereby leading to O 2- Temporary accumulation. In summary, Zn-CDs can slightly induce reactive oxygen species signaling under normal growth conditions, while significantly alleviating oxidative damage under salt stress conditions, thus effectively improving the salt tolerance of honeysuckle berries.

[0079] like Figure 15 As shown, under the same conditions, the activities of SOD, POD, and CAT in honeysuckle berry plants sprayed with Zn-CDs were significantly higher than those in the untreated group. Specifically, under salt stress, the activities of SOD, POD, and CAT in Zn-CDs-treated plants increased by 73.6%, 14.8%, and 10.5%, respectively, compared to the control group; under no stress conditions, Zn-CDs treatment also significantly increased the activities of SOD (19.8%), POD (20.7%), and CAT (28.4%). These results indicate that Zn-CDs treatment increased the activities of related antioxidant enzymes, suggesting a link to enhanced antioxidant defense.

[0080] 2.2.6 Screening of genes related to key pathways in honeysuckle fruit after Zn-CDs application under salt stress Screening results of genes related to key metabolic pathways in honeysuckle fruit after applying Zn-CDs under salt stress showed that ( Figure 16 Zn-CDs primarily affect gene expression in four major functional modules: signal transduction, secondary metabolism, photosynthesis, and the antioxidant system. In signal transduction pathways, differentially expressed genes (DEGs) are mainly enriched in the MAPK signaling pathway – plant and plant hormone signal transduction; secondary metabolism-related DEGs are concentrated in the cutin, suberine and wax biosynthesis, and phenylpropanoid biosynthesis pathways; photosynthesis-related DEGs show significant changes in expression in the oxalative phosphorylation and photosynthesis pathways; and antioxidant system-related genes are mainly enriched in the glutathione metabolism pathway. Notably, under NaCl treatment alone, most DEGs in these pathways show a significant downregulation trend. However, after applying Zn-CDs, the expression levels of these genes are significantly upregulated, suggesting that these pathway changes may be related to Zn-CDs alleviating salt stress damage.

[0081] 2.2.7 qRT-PCR Validation To verify the reliability of the transcriptome analysis data, nine annotated single genes were selected for qRT-PCR analysis. These genes are closely related to pathways such as plant hormone signal transduction, Starch and sucrose metabolism, MAPK signaling pathway, Glycerolipid metabolism, and Glutathione metabolism. After validation, the expression patterns of the nine genes showed ( Figure 17 The expression trends of qRT-PCR results and transcriptome results are basically consistent, indicating that the differential expression results of transcriptome sequencing analysis have high reliability.

[0082] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

Claims

1. A zinc-doped carbon quantum dot from orange peel for alleviating abiotic stress in honeysuckle berries, characterized in that, The orange peel zinc-doped carbon quantum dots are obtained by hydrothermal carbonization using orange peel powder and zinc chloride as raw materials.

2. The orange peel zinc-doped carbon quantum dot according to claim 1, characterized in that, The mass ratio of orange peel powder to zinc chloride is 4~5:

3.

3. The orange peel zinc-doped carbon quantum dots according to claim 2, characterized in that, The solvent for the hydrothermal method is water, and the mass-to-volume ratio of the orange peel powder to water is 4~5g:600~800mL.

4. The orange peel zinc-doped carbon quantum dots according to claim 3, characterized in that, Hydrothermal carbonization involves a hydrothermal reaction at 190-210℃ for 10-12 hours.

5. The orange peel zinc-doped carbon quantum dots according to claim 4, characterized in that, After the hydrothermal reaction is completed, impurities are removed by filtration, and then purified by dialysis to obtain the orange peel zinc-doped carbon quantum dots.

6. The orange peel zinc-doped carbon quantum dot according to claim 5, characterized in that, The molecular weight cutoff for the dialysis is 900 Da to 1100 Da.

7. The orange peel zinc-doped carbon quantum dot according to claim 6, characterized in that, The orange peel zinc-doped carbon quantum dots have a particle size range of 2.13-3.36 nm.

8. The application of the zinc-doped carbon quantum dots from orange peel according to any one of claims 1 to 7 in alleviating abiotic stress in honeysuckle berries, characterized in that, The abiotic stresses are salt stress and / or cadmium stress.

9. The application according to claim 8, characterized in that, A solution of zinc-doped carbon quantum dots from orange peel with a concentration of 800-1200 mg / L was applied to honeysuckle berry plants under salt stress and / or cadmium stress by foliar spraying.

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